A metabolically engineered Escherichia coli and its application in fermentation to produce tyrosol

Abstract

The present invention uses Escherichia coli W3110 as a chassis cell and obtains an L-tyrosine high-yielding Escherichia coli WTY8 strain through metabolic modification. The aromatic aldehyde synthase gene derived from Arabidopsis thaliana is expressed in the WTY8 strain, so that tyrosol can be synthesized from scratch in Escherichia coli using glucose as a substrate, which has low cost and high yield, and is conducive to industrial production.

Description

Technical Field

[0001] The invention relates to a method for preparing tyrosol by fermentation of Escherichia coli through metabolic engineering, belonging to the field of biotechnology. Background Art

[0002] Tyrosol (4-hydroxyphenylethanol, Tyrosol, C 8 H 10 O 2 ) is a natural polyphenol and a derivative of phenylethanol. It is mainly found in Ligustrum lucidum and olive oil, and has multiple pharmacological effects such as antioxidant, anti-cancer, anti-depressant, anti-osteoporosis, anti-inflammatory, cardioprotective and neuroprotective. Its demand is increasing day by day. Currently, three main different methods have been used to produce tyrosol, including direct extraction from plants or cell tissue cultures, chemical synthesis, and microbial synthesis. The abundance of tyrosol in plant sources is low, and the extraction process is complicated, resulting in relatively low production capacity and high cost. On the other hand, chemical methods require relatively extreme reaction conditions and may cause serious environmental pollution. The microbial method of synthesizing tyrosol has more advantages because of its short cycle and easy regulation. Summary of the invention

[0003] The purpose of the present invention is to provide a plasmid-free high-yield L-tyrosine strain obtained by metabolic modification, and based on this, a genetically engineered bacterium capable of producing tyrosol is obtained by recombining exogenous genes. The genetically engineered bacterium can produce tyrosol in high yield.

[0004] The first object of the present invention is to provide a recombinant Escherichia coli for producing tyrosine, wherein the recombinant Escherichia coli W3110 is used as the base bacteria, and the high-L-tyrosine-producing Escherichia coli WTY8 strain is obtained through metabolic modification. The Escherichia coli WTY8 is then used as the host bacteria to express the aromatic aldehyde synthase (PcAAS) gene derived from parsley on the recombinant plasmid pTrc99a, thereby obtaining a tyrosol-producing strain.

[0005] The second object of the present invention is to provide a method for producing tyrosol by fermenting a tyrosol-producing recombinant strain.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a genetically engineered bacterium WTY8, wherein the genetically engineered bacterium WTY8 is obtained by performing the following transformations on Escherichia coli W3110: knocking out the tyrR gene, the pykF gene, the trpD gene and the pheA gene; replacing the promoter of the galP gene, the promoter of the glk gene, the promoter of the ppsA gene and the promoter of the tyrA gene with the trc promoter, respectively; mutating the tyrA gene encoding the amino acid sequence shown in SEQ ID NO: 3 into the tyrA gene encoding the amino acid sequence shown in SEQ ID NO: 4. fbr Gene. (That is, the 53rd amino acid of tyrA protein is mutated from methionine to isoleucine, and the 354th amino acid of tyrA protein is mutated from alanine to valine).

[0008] In one embodiment of the present invention, Escherichia coli W3110 was transformed using CRISPR / Cas 9 technology to obtain a tyrosine high-producing strain WTY8, and the recombinant plasmid was transformed into the WTY8 strain to obtain a tyrosol-producing strain.

[0009] In a second aspect, the present invention also provides an application of the genetically engineered bacteria WTY8 in the production of tyrosine, which can be produced in a laboratory shake flask or in a factory using a fermenter.

[0010] In one embodiment of the present case, the application is:

[0011] The genetically engineered bacteria WTY8 is inoculated into an LB liquid culture medium, and cultured at 37° C. and 180 rpm for 12 hours to obtain a seed solution; the seed solution is inoculated into a fresh LB liquid culture medium at a volume inoculation amount of 1%, and cultured at 37° C. and 180 rpm; when OD600=0.8, IPTG is added with a final concentration of 0.1 mM, and induced culture is performed at 20° C. and 180 rpm for 14 hours; the induced bacterial solution is centrifuged (4000 rpm, 5 minutes), and the obtained wet bacterial bodies are transferred into a fermentation medium, and cultured at 30° C. and 180 rpm for 48 hours to obtain L-tyrosine.

[0012] The fermentation medium includes glucose.

[0013] In one embodiment of the present invention, the volume ratio of the fresh LB liquid culture medium to the fermentation medium is 1:1-2.

[0014] In a third aspect, the present invention further provides an application of the genetically engineered bacterium WTY8 in producing tyrosol, wherein the application is: introducing a recombinant expression plasmid comprising a Trc promoter and an aromatic aldehyde synthase gene into the genetically engineered bacterium WTY8, and the obtained WTYS1 strain produces tyrosol through fermentation culture.

[0015] This gene is used to convert tyrosine into tyrosol.

[0016] Furthermore, the amino acid sequence of the aromatic aldehyde synthase (GenBank: AAA33860.1) is shown in SEQ ID NO:2, and further, the nucleotide sequence is shown in SEQ ID NO:1.

[0017] The vector of the recombinant expression plasmid can be any conventional vector suitable for E. coli expression system in the art, and in one embodiment of the present invention, it is pTrc99a.

[0018] Specifically, in one embodiment of the present invention, the recombinant expression plasmid is obtained by constructing the gene fragment shown in SEQ ID NO: 1 between the PstⅠ and DraⅢ restriction nuclease cutting sites of the pTrc99a vector.

[0019] Specifically, the present invention provides two application methods, a shake flask and a fermenter. The first application (shake flask) is: the recombinant expression plasmid is transferred into the genetically engineered bacteria WTY8, and the positive clone obtained by plate screening is the genetically engineered bacteria WTYS1; the genetically engineered bacteria WTYS1 is inoculated on an LB solid medium containing an antibiotic corresponding to the resistance of the expression plasmid to obtain a single colony,

[0020] A single colony was picked and inoculated into an LB liquid culture medium containing an antibiotic corresponding to the resistance of the expression plasmid, and cultured at 37° C. and 180 rpm for 12 h to obtain a seed solution, the seed solution was inoculated with a fresh LB liquid culture medium containing antibiotics at a volume inoculation amount of 1%, and cultured at 37° C. and 180 rpm until OD600 = 0.8, IPTG with a final concentration of 0.1 mM was added, and induced culture was carried out at 20° C. for 14 h, and the bacteria were collected by centrifugation. The obtained wet bacteria were transferred to a fermentation medium and fermented at 30° C. and 180 rpm for 48 h to produce tyrosol.

[0021] Furthermore, the final concentration of the fermentation medium is composed of: glucose 20g / L, yeast extract 2g / L, KH 2 PO 4 1g / L, MgSO 4 7H 2 O 0.5 g / L, (NH 4 ) 2 SO 4 16g / L, CaCO 3 5g / L, MnSO 4 0.01g / L, FeSO 4 0.01g / L, solvent is water, pH is natural.

[0022] The recombinant Escherichia coli is used to produce tyrosol by fermentation.

[0023] It can be produced in shake flasks in the laboratory or in fermenters in the factory.

[0024] In one embodiment of the present case, the application is (fermentation conditions in a fermenter): the recombinant expression plasmid is transferred into the genetically engineered bacteria WTY8, and the positive clone obtained by plate screening is the genetically engineered bacteria WTYS1; the genetically engineered bacteria WTYS1 is inoculated into an LB liquid culture medium containing an antibiotic corresponding to the resistance of the expression plasmid, and cultured at 37°C and 180rpm for 14h to obtain a first-stage seed solution, which is inoculated into a fresh LB liquid culture medium containing an antibiotic corresponding to the resistance of the expression plasmid at a volume inoculation amount of 1%, and cultured at 37°C and 180rpm for 14h. The obtained second-stage seed solution is transferred into a fermenter filled with fermentation medium at a volume inoculation amount of 10%, with a ventilation volume of 1VVM, and cultured at 37°C. When OD600 reaches 10, IPTG with a final concentration of 0.1mM is added, and glucose with a final concentration of 20g / L is added after induction culture at 20°C for 14h, and fermented at 30°C for 100h. Glucose and yeast powder are added during the fermentation process to maintain the glucose concentration at 5-10g / L.

[0025] The fermentation medium: tryptone 15g / L, yeast extract 5g / L, Na 2 HPO 4 12H 2 O 15.12g / L, KH 2 PO 4 3g / L, MgSO 4 7H 2 O 0.5 g / L, CaCl 2 0.011g / L, NH 4 Cl 1g / L, solvent is water, pH is natural.

[0026] Fermentation tank glucose feed: glucose 600g / L, yeast extract 50g / L.

[0027] Compared with the prior art, the beneficial effects of the present invention are that tyrosol has multiple pharmacological effects such as anti-oxidation, anti-cancer, anti-depression, anti-osteoporosis, anti-inflammatory, cardioprotection and neuroprotection. The present invention uses Escherichia coli W3110 as a chassis cell and obtains an Escherichia coli WTY8 strain with high L-tyrosine production through metabolic modification. The aromatic aldehyde synthase (PcAAS) gene derived from Arabidopsis thaliana is expressed in the WTY8 strain, so that tyrosol can be synthesized from scratch in Escherichia coli using glucose as a substrate, which has low cost and high yield, and is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The plasmid map required for the experiment

[0029] Figure 2 Tyrosine production in E. coli strains WTY1-WTY8

[0030] Figure 3 Tyrosol production in fermentation tanks of Escherichia coli WTYS1 strain DETAILED DESCRIPTION

[0031] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings.

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0033] The culture medium involved in the following examples is as follows:

[0034] LB liquid culture medium formula: yeast powder 5g / L, peptone 10g / L, NaCl 10g / L, solid culture medium plus 1.5%-2.0% agar powder.

[0035] Shake flask fermentation medium: glucose 20g / L, yeast extract 2g / L, KH 2 PO 4 1g / L, MgSO 4 7H 2 O0.5g / L, (NH 4 ) 2 SO 4 16g / L, CaCO 3 5g / L, MnSO 4 0.01g / L, FeSO 4 0.01g / L.

[0036] Fermentation medium: tryptone 15g / L, yeast extract 5g / L, Na 2 HPO 4 12H 2 O 15.12g / L, KH 2 PO 4 3g / L, MgSO 4 7H 2 O 0.5 g / L, CaCl 2 0.011g / L, NH 4 Cl 1g / L.

[0037] Fermentation tank glucose feed: glucose 600g / L, yeast extract 50g / L.

[0038] The tyrosine and tyrosol detection method adopts high performance liquid chromatography (HPLC) detection.

[0039] The specific conditions for tyrosine chromatography detection are as follows: Welch C18 column C18 column (250 mm × 4.6 mm, 5 μm), mobile phase 0.1 mol / L sodium acetate (pH 4.0 ± 0.05)-methanol (90:10), flow rate 1.0 mL / min. UV detector detection wavelength 280 nm, column temperature 30 ° C, injection volume 10 μL.

[0040] The specific conditions for the tyrosol chromatography detection are as follows: Welch C18 chromatographic column (C18 column (250 mm×4.6 mm, 5 μm); mobile phase: acetonitrile: methanol: water: acetic acid = 36:6:57.98:0.02; flow rate 1.0 mL / min; UV detector detection wavelength 205 nm; column temperature 30°C, injection volume 10 μL.

[0041] The present invention will be further described below in conjunction with specific implementation cases.

[0042] The invention obtains a tyrosine high-yield strain by gene editing, and the detailed process is as follows: gene editing is performed on Escherichia coli W3110 by CRISPR / Cas9 technology, tyrR, pykF, trpD and pheA genes are knocked out in sequence to obtain WTY1, WTY2, WTY3 and WTY4 strains respectively, and then gene overexpression is performed on the basis of the WTY4 strain, and the original promoters of galP, glk and ppsA genes are replaced with trc promoters in sequence to obtain WTY5, WTY6 and WTY7 strains respectively; the tyrA gene (encoding an amino acid sequence as shown in SEQ ID NO: 3 and a nucleotide sequence as shown in SEQ ID NO: 5) and its original promoter are replaced with tyrA fbr The gene (encoding the amino acid sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 6) and the trc promoter were used to obtain the high-tyrosine-producing Escherichia coli WTY8.

[0043] Example 1: CRISPR / Cas 9 gene knockout operation, taking the tyrR gene as an example.

[0044] (1) Preparation of E. coli W3110 / pCas9 competent cells

[0045] 50 ng of pCas plasmid was transformed into Escherichia coli W3110 competent cells by heat shock at 42°C for 90 seconds, applied to solid LB medium (100 mg / L kana) and cultured in an incubator at 30°C overnight to obtain Escherichia coli W3110 / pCas strain. A single colony was picked and inoculated into 5 mL LB test tube medium (100 mg / L kana), and 50 μL 1 M L-arabinose (L-Ara) solution was added, and cultured overnight at 30°C, 180 rpm to obtain seed solution. Take 500 μL of seed solution and inoculate it into 50 mL of LB medium (100 mg / L kana), add 500 μL of 1M L-Ara solution, culture at 30°C, 180 rpm until OD600 = 0.4-0.6, ice bath for 5 min, pour the bacterial solution into a 50 mL sterile centrifuge tube on a clean bench, centrifuge at 8°C, 5000 rpm for 10 min, discard the supernatant on a clean bench, add 20 mL of pre-cooled 100 mM CaCl 2 Resuspend the cells and place on ice for 30 min. Centrifuge at 8°C, 5000 rpm for 10 min. Discard the supernatant aseptically and add 0.5 mL of pre-cooled 100 mM CaCl 2 solution and 60% glycerol, resuspend the cells, dispense 50 μL each into sterile 1.5 mL EP tubes, and store at -80°C.

[0046] (2) Donor DNA preparation

[0047] Obtain the upstream homology arm fragment, amplification system: 1 μL of 50 ng / μL E. coli W3110 genomic DNA as template, 1 μL of 10 μM primers tyrR-F1 and tyrR-R1 (Table 1), 2× PrimeSTAR MAX Premix 25 μL, ddH 2 O22μL. PCR reaction conditions were: pre-denaturation at 98℃ for 5min, followed by temperature cycling at 98℃ for 10sec; 57℃ for 15sec; 72℃ for 1min; 30 cycles in total, with a termination temperature of 4℃. After PCR amplification was completed, the correct PCR product was verified by 1% agarose gel electrophoresis. The upstream homology arm fragment was purified using PCR Purification Kit.

[0048] The downstream homology arm fragment was obtained by referring to the upstream homology arm fragment, and the primers were tyrR-F2 / tyrR-R2 (Table 1). Overlap PCR was used to obtain DonorDNA, and the amplification system was: 1 μL of the upstream and downstream homology arm fragments at 50 ng / μL as templates, 1 μL of 10 μM primers tyrR-F1 and tyrR-R2, 2× PrimeSTAR MAX Premix 25 μL, ddH2 O 21μL. PCR reaction conditions were: pre-denaturation at 98°C for 5 min, followed by temperature cycling at 98°C for 10 sec; 57°C for 15 sec; 72°C for 1 min; 30 cycles in total, with a termination temperature of 4°C.

[0049] (3) SgRNA preparation

[0050] According to the principle of CRISPR / Cas 9 gene editing, site-directed mutagenesis primers were designed to mutate the original SgRNA into a specific guide SgRNA sequence. Amplification system: 1 μL 50 ng / μL PTarget-F plasmid DNA (Addgene Plasmid #62226) as template, 1 μL of primers pTar(tyrR)-F and pTar(tyrR)-R) at a concentration of 10 μM, 2× PrimeSTAR MAXPremix 25 μL, ddH 2 O 22μL. PCR reaction conditions were: pre-denaturation at 98℃ for 5min, followed by temperature cycling at 98℃ for 10sec; 57℃ for 15sec; 72℃ for 2min; 30 cycles in total, with a termination temperature of 4℃. After PCR, the correct PCR product was verified by 1% agarose gel electrophoresis and used PCR Purification Kit was used to recover and purify the PCR system to 30 μL. The purified fragments were digested with the template DNA using the Dpn I restriction enzyme system. The restriction enzyme system was: QuickCut Dpn I 1 μL, 10× buffer 3 μL, and the reaction was carried out at 37°C for 2.5 h. After the reaction, the fragments were used Purify and recover the product to 20 μL using PCR Purification Kit. Take 5 μL of the purified product to transform E. coli DH5a competent cells, screen using LB plates (50 mg / L spectinomycin hydrochloride (SD)) and sequence verification. Inoculate the correctly mutated strain into 10 mL LB liquid medium (50 mg / L SD), culture at 37°C and 180 rpm for 12 h, obtain bacterial solution, extract the plasmid and record it as pTarget-tyrR, and store it at -20°C.

[0051] (4) Gene editing operations

[0052] Take competent cells carrying pCas plasmid from -80℃, let stand in ice bath for 2min, add 500ng Donor DNA and 50ng pTarget-tyrR, mix gently and let stand on ice for 30min. After standing, heat shock at 42℃ for 90s and let stand in ice bath for 2min. After standing, add 1mL (ice bath) of LB medium to the competent cells and place in shaker at 30℃, incubate at 180rpm for 2h. After incubation, take 100μL and apply it to LB solid medium (50mg / LSD+100mg / L kana) and culture overnight at 30℃.

[0053] Several single clones were picked from the transformation plate as templates and verified by colony PCR using tyrR-TF / tyrR-TR as primers. Positive clones were used to further eliminate the pTarget-tyrR plasmid.

[0054] (5) Elimination of pTarget-tyrR plasmid

[0055] The positive clones were inoculated into 5 mL LB liquid medium (100 mg / L kana), IPTG with a final concentration of 0.1 mM was added to induce the pCas plasmid to eliminate the pTarget-tyrR plasmid, and cultured at 30°C, 180 rpm for 16 h, and then streaked on LB solid medium (100 mg / L kana) and cultured overnight at 30°C. Single colonies were picked (here only half of the size of the single colony was picked) and numbered, and inoculated into the corresponding numbered area on LB solid medium (50 mg / LSD) according to the number, and cultured overnight at 30°C. Single colonies that cannot grow in the corresponding area of ​​LB solid medium (50 mg / L SD) are strains that have successfully eliminated pTarget-tyrR.

[0056] (6) pCas plasmid elimination

[0057] After all gene editing is completed, the pCas plasmid is eliminated and the strain construction is completed for subsequent fermentation culture and other experiments.

[0058] The single colony successfully eliminated from pTarget-tyrR was inoculated into 5mL LB medium and cultured at 37°C and 180rpm for 16h. Take 10μL of the cultured bacterial solution and streak it on LB solid medium and culture it at 37°C overnight. After the culture is completed, the streaked single colony is numbered, and the numbered single colony is picked (here only half of the size of the single colony is picked) and inoculated into the corresponding area on LB solid medium (100mg / Lkana), and cultured overnight at 30°C. The single colony that cannot grow in the corresponding area of ​​LB solid medium (100mg / L kana) is the pCas successfully eliminated strain. The pCas eliminated strain is the engineered strain that has completed the editing. It is inoculated into LB liquid medium and cultured overnight at 37°C and 180rpm shaking, and the strain is stored at -80°C for subsequent experimental operations.

[0059] 3. If the next round of gene editing is performed, the pTarget-tyrR strain that has been successfully eliminated will be prepared again as a competent cell carrying the pCas plasmid. If the subsequent gene editing is gene knockout, refer to the tyrR gene knockout operation process, the difference is that the corresponding primers in Table 1 are used, and the operation is performed according to the numbers corresponding to the primers in Table 1 and the above gene knockout operation.

[0060] 4. The operation of CRISPR-Cas 9 replacing gene promoter, taking galP gene as an example.

[0061] Replacement of the galP original promoter is a gene replacement. Compared with tyrR gene knockout, the difference is that the trc promoter fragment is used as a template in the DonorDNA construction step, and galP-F3 / galP-R3 (Table 1) is used as primers. The amplification system is: 1 μL of 50 ng / μL pTrc99a plasmid as a template, 1 μL of primers galP-F3 and galP-R3 at a concentration of 10 μM, 2× PrimeSTAR MAX Premix 25 μL, ddH 2 O 22μL. PCR reaction conditions were: pre-denaturation at 98°C for 5 min, followed by temperature cycling at 98°C for 10 sec; 57°C for 15 sec; 72°C for 1 min; 30 cycles in total, with a termination temperature of 4°C.

[0062] The upstream and downstream homology arm fragments were obtained by amplification using galP-F1 / galP-R1 and galP-F2 / galP-R2 (Table 1) as primers, respectively, and Escherichia coli W3110 genomic DNA as a template; DonorDNA was constructed using 50 ng / μL of the upstream and downstream homology arms and 1 μL of the trc promoter fragments as templates, 10 μM galP-F1 and galP-R2 as primers, 2× PrimeSTAR MAX Premix 25 μL, ddH 2O 21μL, and fuse the three fragments into the desired DonorDNA by Overlap PCR. The PCR program refers to the tyrR gene.

[0063] 5. CRISPR-Cas 9 operation on tyrA anti-feedback inhibition point mutation and promoter replacement.

[0064] TyrA anti-feedback inhibition point mutation and replacement promoter belong to gene replacement. First, the anti-feedback inhibition target gene tyrA fbr The construction of tyrA sequence was carried out by mutating the amino acid at position 53 from methionine to isoleucine and the amino acid at position 354 from alanine to valine. The specific process was as follows: first, the tyrA1 fragment was amplified, and the amplification system was as follows: 1 μL of 50 ng / μL E. coli W3110 genomic DNA as template, 1 μL of 10 μM primers tyrA-F3 and tyrA-R3 (Table 1), 25 μL of 2× PrimeSTAR MAX Premix, and ddH 2 O 22μL. PCR reaction conditions were: pre-denaturation at 98℃ for 5min, followed by temperature cycling at 98℃ for 10sec; 57℃ for 15sec; 72℃ for 1min; 30 cycles in total, with a termination temperature of 4℃. After PCR amplification, the correct PCR product was verified by 1% agarose gel electrophoresis. The tyrA1 fragment was purified by PCR Purification Kit. The tyrA2 fragment was obtained by referring to the tyrA1 fragment, and the primers were tyrA-F4 / tyrA-R4. fbr The upstream and downstream homology arm fragments were amplified using tyrA-F1 and tyrA-R1, tyrA-F2 and tyrA-R2 as primers, and Escherichia coli W3110 genomic DNA as template. Amplification system: 1 μL of 50 ng / μL upstream and downstream homology arm fragments as templates, 1 μL of 10 μM primers tyrA-F3 and tyrA-R4, 2× PrimeSTAR MAX Premix 25 μL, ddH 2 O 21μL. PCR reaction conditions were: pre-denaturation at 98℃ for 5min, followed by temperature cycling at 98℃ for 10sec; 57℃ for 15sec; 72℃ for 1min; 30 cycles in total, with a termination temperature of 4℃. The correct PCR product was verified by 1% agarose gel electrophoresis. Purify the target gene tyrA using PCR Purification Kit fbr Then, the target gene tyrA fbrWith the upstream and downstream homology arm fragments as templates and tyrA-F1 / tyrA-R2 (Table 1) as primers, Overlap PCR was used to fuse the three fragments into the desired DonorDNA. The remaining steps refer to tyrR gene knockout.

[0065] Table 1: Gene editing primers

[0066]

[0067]

[0068]

[0069]

[0070] 4. Construction of tyrosol-producing strains

[0071] The PcAAS target gene was optimized and synthesized by Beijing Qingke Biotechnology Co., Ltd. and constructed between the PstⅠ and DraⅢ restriction nuclease sites of the pTrc99a vector. The plasmid was transformed into the WTY8 competent cell to obtain the WTYS1 strain that produces tyrosol.

[0072] Example 2: Production of tyrosine and tyrosol by shake flask fermentation

[0073] (1) The metabolically modified Escherichia coli strains WTY1 to WTY8 prepared in Example 1 were inoculated into 50 mL of antibiotic-free LB liquid culture medium, respectively, and cultured at 37° C. and 180 rpm for 12 h to prepare seed solution.

[0074] (2) The seed solution prepared in step (1) was inoculated into 50 mL of antibiotic-free LB liquid culture medium at a volume ratio of 1%, and cultured at 37° C. and 180 rpm. When OD600 = 0.8, IPTG was added at a final concentration of 0.1 mM, and induced culture was performed at 20° C. and 180 rpm for 14 h.

[0075] (3) The induced bacterial solution obtained in step (2) was centrifuged at 4000 rpm for 5 min to collect the bacterial cells, and the cells were resuspended in 3 mL of fermentation medium and transferred to 50 mL of fermentation medium and cultured at 30°C and 180 rpm for 48 h.

[0076] After 48 hours, samples were taken and the L-tyrosine content was tested by HPLC. The tyrosine production results of E. coli strains WTY1-WTY8 were as follows: Figure 2 As shown, the WTY8 strain produced the highest amount of tyrosine, which was 2.35 g / L.

[0077] (3) From the test results, it is known that the WTY8 strain produces the highest amount of tyrosine. The WTY8 strain was made into a competent state for transformation, and the recombinant plasmid pTrc99a-PcAAS was transformed to obtain the WTYS1 strain to produce tyrosol. The WTYS1 strain was inoculated with 1% inoculum in 50mL of medium containing Amp (100mg / L), and cultured at 37°C and 180rpm until OD600=0.8, and then IPTG with a final concentration of 0.1mM was added for overnight induction at 20°C. Then the cells were collected and transferred to 50mL of fermentation medium at 30°C and 180rpm for 48h. Samples were taken at 48h and the product content was detected by HPLC. The WTS1 strain was obtained according to the same steps as above, except that the WTY8 strain was replaced by E.coli W3110. The tyrosol production results are shown in Table 3.

[0078] Table 3 Tyrosol production by shake flask fermentation of Escherichia coli strains WTS1 and WTYS1.

[0079]

[0080] Example 3: Production of tyrosol by fermentation in a fermenter

[0081] (1) The prepared Escherichia coli WTYS1 strain was inoculated into 50 mL LB liquid medium (100 mg / LAmp) and cultured at 37° C. and 180 rpm for 12 h to prepare the first stage seed solution;

[0082] (2) The seed solution prepared in step (1) was inoculated at a volume ratio of 1% into 100 mL LB liquid culture medium (100 mg / L Amp), and cultured at 37° C. and 180 rpm for 12 h to obtain a second-stage seed solution; (3) The second-stage seed solution prepared in step (2) was transferred at a volume ratio of 10% into a 5-L fermentation tank containing 1.8 L fermentation medium (100 mg / L Amp), and cultured at a temperature of 37° C., aeration ratio of 1 VVM, dissolved oxygen of 40%, and pH = 7.0 until OD 600 When the pH value reaches 10-11, IPTG with a final concentration of 0.1 mM is added, and the mixture is induced and cultured at 20°C for 16 hours, and then fermented at 30°C for 100 hours to obtain a fermentation liquid containing tyrosol. 5M ammonia water is added at 30°C to adjust the pH; after induced culture for 16 hours, the glucose is controlled at 5-10 g / L by adding 600 g / L glucose.

[0083] After induction, samples were taken every 4 hours to test OD600 and tyrosol production, and the samples were tested by HPLC. Figure 3As shown: the tyrosol yield of strain WTYS1 in a 5L fermenter after fermentation for 100h is 6.2g / L. The engineered strain constructed by the present invention and the fermentation strategy used provide guidance for industrial green and efficient production of tyrosol.

[0084] Although the present invention has been disclosed as above in the 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.

[0085] SEQ ID NO:1

[0086]

[0087] SEQ ID NO:2

[0088] MGSIDNLTEKLASQFPMNTLEPEEFRRQGHMMIDFLADYYRKVENYPVRSQVSPGYLREILPESAPYNPESLETILQDVQTKIIPGITHWQSPNFFAYFPSSGSTAGFLGEMLSTGFNVVGFNWMVSPAATELENVVTDWFGKMLQLPKSFLFSGGGGGVLQGTTCEAILCTLVAARDKNLRQHGMDNIGKLVVYCSDQTHSALQKAAKIAGIDPKNFRAIETTKSSNFQLCPKRLESAILHDLQNGLIPLYLCATVGTTSSTTVDPLPALTEVAKKYDLWVHVDAAYAGSACICPEFRQYLDGVENADSFSLNAHKWFLTTLDCCCLWVRNPSALIKSLSTYPEFLKNNASETNKVVDYKDWQIMLSRRFRALKLWFVLRSYGVGQLREFIRGHVGMAKYFEGLVNMDKRFEVVAPRLFSMVCFRIKPSAMIGKNDEDEVNEINRKLLESVNDSGRIYVSHTVLGGIYVIRFAIGGTLTDINHVSAAWKVLQDHAGALLDDTFTSNKLVEVLS

[0089] SEQ ID NO:3

[0090] MVAELTALRDQIDEVDKALLNLLAKRLELVAEVGEVKSRFGLPIYVPEREASMLASRRAEAEALGVPPDLIEDVLRRVMRESYSSENDKGFKTLCPSLRPVVIVGGGGQMGRLFEKMLTLSGYQVRILEQHDWDRAADIVADAGMVIVSVPIHVTEQVIGKLPPLPKDCILVDLASVKNGPLQAMLVAHDGPVLGLHPMFGPDSGSLAKQVVVWCDGRKPEAYQWFLEQIQVWGARLHRISAVEHDQNMAFIQALRHFATFAYGLHLAEENVQLEQLLALSSPIYRLELAMVGRLFAQDPQLYADIIMSSERNLALIKRYYKRFGEAIELLEQGDKQAFIDSFRKVEHWFGDYAQRFQSESRVLLRQANDNRQ

[0091] SEQ ID NO:4

[0092] MVAELTALRDQIDEVDKALLNLLAKRLELVAEVGEVKSRFGLPIYVPEREASILASRRAEAEALGVPPDLIEDVLRRVMRESYSSENDKGFKTLCPSLRPVVIVGGGGQMGRLFEKMLTLSGYQVRILEQHDWDRAADIVADAGMVIVSVPIHVTEQVIGKLPPLPKDCILVDLASVKNGPLQAMLVAHDGPVLGLHPMFGPDSGSLAKQVVVWCDGRKPEAYQWFLEQIQVWGARLHRISAVEHDQNMAFIQALRHFATFAYGLHLAEENVQLEQLLALSSPIYRLELAMVGRLFAQDPQLYADIIMSSERNLALIKRYYKRFGEAIELLEQGDKQAFIDSFRKVEHWFGDYVQRFQSESRVLLRQANDNRQ

[0093] SEQ ID NO:5

[0094]

[0095] SEQ ID NO:6

[0096]

Claims

1. An application of a genetically engineered bacterium WTYS1 in producing tyrosol, wherein the genetically engineered bacterium WTYS1 is obtained by performing the following transformations on Escherichia coli W3110: knocking out the tyrR gene, the pykF gene, the trpD gene and the pheA gene; replacing the promoters of the galP gene, the glk gene, the ppsA gene and the tyrA gene with the trc promoter respectively; mutating the tyrA gene encoding the amino acid sequence shown in SEQ ID NO:3 into the tyrAfbr gene encoding the amino acid sequence shown in SEQ ID NO:4; obtaining the genetically engineered bacterium WTY8, introducing a recombinant expression plasmid containing the Trc promoter and an aromatic aldehyde synthase gene into the genetically engineered bacterium WTY8 to obtain the genetically engineered bacterium WTYS1, and the obtained genetically engineered bacterium WTYS1 produces tyrosol through fermentation and culture; the amino acid sequence of the aromatic aldehyde synthase is shown in SEQ ID NO:2; and the vector of the recombinant expression plasmid is pTrc99a.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the aromatic aldehyde synthase is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that: The recombinant expression plasmid was transferred into the genetically engineered bacteria WTY8, and the positive clone obtained by plate screening was the genetically engineered bacteria WTYS1; the genetically engineered bacteria WTYS1 was inoculated into an LB liquid culture medium containing an antibiotic corresponding to the resistance of the recombinant expression plasmid, and cultured at 37°C and 180 rpm for 14 h to obtain a first-stage seed solution, and the first-stage seed solution was inoculated into a fresh LB liquid culture medium containing an antibiotic corresponding to the resistance of the recombinant expression plasmid at a volume inoculation of 1%, and cultured at 37°C and 180 rpm for 14 h to obtain a second-stage seed solution, and the obtained second-stage seed solution was transferred into a fermentation tank filled with fermentation medium at a volume inoculation of 10%, with a ventilation volume of 1VVM, and cultured at 37°C. When OD600 reached 10, IPTG with a final concentration of 0.1 mM was added, and glucose with a final concentration of 20 g / L was added after induction culture at 20°C for 14 h, and fermented at 30°C for 100 h. Glucose and yeast powder were added during the fermentation process to maintain the glucose concentration at 5-10 g / L.

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